In 1993, six novel galactosyl ceramides with unique α-glycosidic linkages were isolated from the marine sponge Agelas mauritianus near Okinawa, Japan. (Natori, T. et al., Tetrahedron Lett., 34, 5591 (1993); Natori, T. et al., Tetrahedron, 50, 2771 (1994).) These compounds showed highly potent anti-tumor activities, which prompted various synthetic studies. (Motoki, K. et al., Bioorg. Med. Chem. Lett., 5, 705 (1995).) Among the many analogues synthesized, KRN7000 (see structure below) was found to be the most potent (Kobayashi, E. et al., Bio. Med. Chem., 4, 615 (1996)) and extensive mechanistic studies indicated that the anti-tumor activity was the result of CD1d-dependent natural killer T-cell (NKT) stimulation. (Kobayashi, E. et al., Bio. Med. Chem., 4, 615 (1996); Kawano, T. et al., Science, 278, 1626 (1997).) CD1d molecule is a member of the CD1 family proteins that present lipid antigens to NKT cells to activate the immune response. It is proposed that CD1d recognizes KRN7000 and the binding complex interacts with the T-cell receptor (TCR) to NKT cells stimulating the release of two major cytokines known as INF-γ and IL-4. (Wu, D. et al., Proc. Natl. Acad. Sci., 102, 1351 (2005); Porcelli, S. A. and Modlin R. L., Annu. Rev. Immunol, 17, 297 (1999); Kinjo, Y. et al., Nature, 434, 520 (2005).) The two cytokines, however, can cancel each other's beneficial therapeutic effect as one pathway down-regulates the other. (Pal, E. et al., J. Immunol., 166, 662 (2001); Berkers, C. R. and Ovaa, H., Trends. Pharmacol. Sci., 26, 252 (2005).) Interestingly, different KRN7000 analogs selectively stimulate cytokine production. For example, OCH (see structure below), which has only 9 carbons in the acyl chain of ceramide, produces predominately IL-4 and exhibits greater efficacy than KRN7000 against the autoimmune disease experimental allergic encephalomyelitis. (Pal, E. et al., J. Immunol., 166, 662 (2001); Miyamoto, K. et al., Nature, 413, 531 (2001).) Whereas, the C-glycoside analogue of KRN7000, cKRN7000 (see structure below) upregulates IFN-γ and is 100 times more potent than KRN7000 in inhibiting tumor growth in mice. (Yang, G. et al., Angew. Chem. Int. Ed, 43, 3818 (2004).)

Numerous efforts have been invested in the syntheses of α-GalCer analogs to access these biologically important compounds in pure form for biological and biomedical studies. However, considerable challenges remain. Arguably, the biggest hurdle in the synthesis is the glycosylation reaction, which often gives low yields and poor α/β selectivity. To achieve the desired chemo- and stereoselectivity, multi-step protections and deprotections are required lowering the overall synthetic efficiency. Initially, glycosyl fluorides (Sakai, T. et al., J. Med. Chem., 41, 650 (1998); Ndonye, R. M. et al., J. Org. Chem., 70, 10260 (2005); Kim, S. et al., Synthesis, 847 (2004); Morita, M. et al., J. Med. Chem., 38, 2176 (1995)) and trichloroacetimidates (Kim, S. et al., Synthesis, 847 (2004); Xia, C. et al., Bioorg. Med. Chem. Lett., 16, 2195 (2006); Plettenburg, O. et al., J. Org. Chem., 67, 4559 (2002); Figueroa-Perez, S, and Schmidt, R. R., Carbohydrate Research, 328, 95 (2000)) were the most commonly used glycosyl donors. These protocols gave marginal yields (30%-60%) and were often complicated by the formation of α/β mixtures. Other glycosyl donors such as bromides (Goff, R. D. et al., J. Am. Chem. Soc., 126, 13602 (2004), thiogalactoside (Plettenburg, O. et al., J. Org. Chem., 67, 4559 (2002)), and phosphites (Luo, S. Y. et al., J. Org. Chem., 71, 1226 (2006)) have also been employed, however neither yields nor stereoselectivities were improved.
Recently, a significant advance in the glycosylation reaction using glycosyl iodide donors was reported. (Du, W. and Gervay-Hague, J., Org. Lett., 7, 2063 (2005).) Reactions of per-O-benzylated galactosyl iodide with an azido sphingosine in the presence of tetrabutylammonium iodide afforded exclusively the α-anomer in over 90% yield. An azido group is used in place of the amide because, if left intact during the glycosylation, the amide deactivates the primary hydroxyl of the acceptor through unfavorable hydrogen bonding interactions. (Polt, R. et al., J. Am. Chem. Soc., 114, 10249 (1992); Schmidt, R. R. and Zimmermann, P., Angew. Chem. Int. Ed., 25, 725 (1986).)
There have been several attempts to incorporate fully functionalized glycolipids as acceptors but with limited success. Ceramide acceptors as such give complex mixtures with per-O-benzylated fluoride and trichloroacetimidate donors; whereas a TBS-protected ceramide acceptor affords coupling products in moderate yields and selectivity (e.g., Fluoride, 68%, α/β=1.7/1; imidate, 63%, only α). (Kim, S. et al., Synthesis, 847 (2004).) Similarly, 3,4,6-tri-O-acetyl, 2-O-benzyl galactosyl bromide had been coupled with acetylated acceptor in 62% yields to obtain α-linked product along with a minor, difficult to separate, β-isomer. (Goff, R. D. et al., J. Am. Chem. Soc., 126, 13602 (2004).)
As discussed above, a major challenge in the synthetic pathway of glycolipids is the glycosylation reaction, which often gives low yields and poor α/β selectivity. Historically, the most common method to synthesize β-linked glycolipids involved the glycosylation between a protected lipid acceptor (usually azido-sphingosine or azido-phytosphingosine) and a protected donor containing a C2 participating group designed to capitalize on anchiomeric assistance. Some common examples from the literature include the use of per-O-benzoylated thiogalactosides (Fukunaga, K. et al., Bioorg. Med. Chem. Lett., 13, 813, (2003)), per-O-acetyl sugars (Morita, M. et al., Bioorg. Med. Chem. Lett., 5, 699, (1995)), and per-O-pivaloylated trichloroacetimidates (Matto, P. et al., J. Org. Chem., 72, 7757, (2007).)
While the stereo-selectivity reported in these reactions highly favors the desired β-glycoside, the presence of a participating group at the C2 position lowers the reactivity of the donor and severely limits the options available when working towards more complex synthetic targets (such as in oligosaccharide synthesis) thereby making several (and often redundant) protection-deprotection steps required. Recently, significant advances in yield and α-selectivity of glycosylation reactions have been achieved via the use glycosyl iodide donors (Du, W., Kulkarni, S. S., Gervay-Hague, J. Chem. Commun., 2336-2338 (2007)). But prior methods of making β-linked glycolipids involved multi-step schemes with isolation after each intermediate step (Lichtenthaler, F. W., Kohler, B. Carbohydrate Research, 258, 77-85 (1994)). What is needed is a single-pot process for preparation of α-linked and β-linked glycolipids. It is an attractive strategy to employ fully functionalized ceramide acceptors because tedious protection and deprotection steps, many subsequent to glycosidation, can be alleviated. Surprisingly, the present invention meets this and other needs.